The invention relates generally to in vitro-differentiated retinal ganglion cells and methods for obtaining the cells, and more particularly to methods for obtaining the cells by exposing a cultured retinal ganglion cell line to a protein kinase inhibitor.
The study of optic neuropathies, e.g. glaucoma, and an involved neuron, the retinal ganglion cell (RGC), has been limited to primary cell culture and in vivo models as it is difficult to use cell culture to study RGC pathophysiology. Levin L A, J. Glaucoma 10:S19-21 (2001). Harvesting retinas and identifying RGCs by retrograde labeling or immunocytochemistry is labor intensive and time-consuming. Leahy K M, et al., Exp. Eye Res. 79:131-140 (2004). In addition, RGC purification using antibodies to cell-surface antigens or anatomical location is a lengthy and frequently low-yield process that results in a heterogeneous cell population. Barres B A, et al., Neuron 1:791-803 (1988); Lindqvist N. et al., Brain Res. Mol. Brain Res. Protoc. 10:75-83 (2002). Furthermore, the cells that arise upon differentiation of neuronal cell lines, e.g. pheochromocytoma-derived PC-12 cells, teratoma-derived NT2-N cells or neuroblastoma-derived SY5Y cells, are not retina-derived and do not share phenotypic properties of RGCs. See Greene L A & Tischler A S, Proc. Natl. Acad. Sci. USA 73:2424-2428 (1976); Pleasure S J, et al., J. Neurosci. 12:1802-1815 (1992); and Biedler J L, et al., Cancer Res. 33:2643-2652 (1973).
RGC-5 (ATCC No. PTA 6600), a retinal ganglion cell line derived by transforming postnatal day one rat retinal cells with ψ2 EIA virus, expresses neuronal markers characteristic of RGCs, e.g. Thy-1, Brn-3c, neuritin, and the NMDA-R1 and GABA-B receptors, but does not express glial fibrillary acidic protein (GFAP), an astrocyte marker. Krishnamoorthy R R, et al., Brain Res. Mol. Brain Res. 86:1-12 (2001), incorporated herein by reference as if set forth in its entirety.
Although RGC-5 cells share antigens with RGCs, RGC-5 cells are significantly different, most significantly in that RGC-5 cells are mitotically active while RGCs are not. Also, cultured RGC-5 cells are morphologically more similar to glial cells than to primary RGCs and do not express the repertoire of ion channels that are characteristic of RGCs. Moorhouse A J, et al., Brain Res. 1003;205-208 (2004). As such, what is needed is an RGC-like cell line having pharmacological, biochemical and electrophysiological characteristics that resembles true RGCs more closely than cell lines such as RGC-5 cells.